Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell migration and signaling by complex, integrated mechanisms. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for efficient cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce normal cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast and confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We previously established that the topography of the extracellular matrix (ECM) plays a vital role in regulating cytoskeletal organization, cell morphology, and cell migration by demonstrating that one-dimensional (1D) micropatterned lines mimic the functions of the fibrillar ECM structures found in three-dimensional cell-derived matrix. We extended these studies to establish the mechanism by which fibrillar topography evokes rapid, efficient cell migration in fibroblasts and how this mode of migration differs from migration studied previously using regular two-dimensional (2D) tissue culture substrates. Current studies are focused on characterizing and comparing the dynamics of cell-matrix adhesions and the matrix itself in 3D collagen gel environments. We are comparing adhesion dynamics and cell migration in collagen gels with different matrix organization, i.e., highly reticular matrix compared to matrices with differing levels of fibrillar structures, which we alter by varying polymerization conditions without changing ligand density. These different types of collagen matrix correspond to different types of extracellular matrix in vivo, and we are documenting differences in cell adhesive and migratory responses. We have also characterized the regulatory and functional crosstalk between actomyosin contractility and microtubule post-translational modification in cell adhesion, migration, matrix assembly, and embryonic organ branching. Our studies have identified a homeostatic balance between actomyosin-mediated contraction and the level of microtubule post-translational acetylation. This balance is mediated by interactions between myosin phosphatase and myosin light chain or, alternatively, the microtubule deacetylase HDAC6. Specifically, the myosin phosphatase target subunit of myosin phosphatase can bind and modulate either non-muscle myosin light chain or the enzyme HDAC6. Its phosphatase activity regulates the activities of these substrates. The balance between contractility and microtubule acetylation controls the maturation of cell-matrix adhesions by regulating integrin endocytosis and cell surface density of the alpha-5/beta-1 integrin. The latter change affects the extent of fibronectin matrix assembly and rates of cell migration. In addition, altering this balance affects the rate of embryonic organ branching. These studies are being extended to evaluate a possible role for microtubule acetylation in modulating the phenotype of salivary gland progenitor cells. This combined knowledge regarding the regulation of cell migration and phenotype should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of the precise manner in which cells move and interact with their matrix environment will also facilitate tissue engineering studies.